Arp2/3 complex, a 224 kDa assembly of 7 proteins, is the central actin nucleation machine in the cell. It controls assembly of actin filament networks that are essential to process ranging from motility to signal transduction to bacterial pathogenesis. The intrinsic actin nucleating activity of Arp2/3 complex is low, but is greatly enhanced through binding of many proteins, including WASp, N-WASp, Scar/WAVE, Bee1 and ActA. Research in the Rosen Proposal is focused on discovering the residues and structures through which activator proteins bind Arp2/3 complex and G-actin to control activity of the assembly. The work extends an existing multi-year collaboration between the Rosen, Pollard and Li groups. NMR analyses of resonance broadening and transferred-NOEs will rapidly discover the specific regions and structures within the VCA segments of a series of activators that contact Arp2/3 complex. Mutagenesis experiments will define the thermodynamic contributions of individual contacts to binding and activation of Arp2t3 complex, and will discover if thermodynamic coupling of VCA binding to binding of F-actin, nucleotides or G-actin is mediated by the C or A regions. NMR and mutagenesis studies will test two rival models for the interaction of full length WASp/N-WASp with Arp2/3 complex to discover whether the B and GBD regions of these proteins modulate activity of Arp2/3 complex through direct binding to the assembly, or indirectly through modulating affinity of the VCA. NMR studies of selectively labeled intact Arp2/3 complex will reveal the surfaces of the ARPC3/p21 and ARPC5/p16 subunits that mediate binding to activators, and will lead to general procedures for mechanistic analysis of very large systems by NMR. Finally, NMR and mutagenesis experiments will define the structural and thermodynamic bases for binding of VCA peptides to G-actin. The combined data will allow common features and key differences among Arp2/3 complex activators to be discerned, and structural and thermodynamic models of activation to be developed. Experiments throughout will only be possible through collaborative exchange of reagents and expertise among the Rosen, Pollard and Li groups. Identification of minimal Arp2/3 complex binding and activation elements in VCA segments and full length WASp will direct crystallographic, radiation footprinting and EM studies in the Pollard/Almo and Hanein/Volkmann Proposals, and aid interpretation of structures determined therein.